The long-term goal of this proposal is to delineate the mechanisms by which amino-acid radicals participate in both productive and destructive redox reactions in living organisms. Controlled radical chemistry occurs in enzymes that use amino acids in catalytic and multistep electron-transfer reactions. Amino-acid radical enzymes are involved in a range of chemical transformations some of which are fundamental to aerobic life on Earth. It is of vital interest to delineate these chemical reactions in great detail for a numbe of reasons. These include, for example, developing anticancer drugs, understanding the interactions of non-steroidal anti- inflammatory drugs (NSAIDs) such as aspirin and ibuprofen with their protein targets, and laying the foundation for sustainable solar energy production. Importantly, there is also a sinister side to amino-acid radicals as these species are generated during oxidative stress conditions and can cause significant cellular damage. Despite the biochemical importance of amino-acid radicals, surprisingly little is known about their fundamental thermodynamic and kinetic properties. It is very challenging to study these species due to their highly oxidizing and reactive nature. As a result, no guide is currently available for comparing the formal reduction potentials of amino-acid radicals and correlating these values with the properties of the surrounding protein. Studies aimed at characterizing the proton-coupled electron transfer (PCET) reactions associated with tyrosine oxidation-reduction are currently of high interest but are largely conducted on small-molecule models free in solution. The correlation between the solution chemistry and the protein chemistry is not straightforward. Furthermore, it is well known that the protein matrix can modulate the lifetime of the amino- acid radical by many orders of magnitude but there is little information on how this occurs. We have developed a protein system that can provide novel and important information about these issues. The a3X constructs are well-structured proteins supporting reversible amino-acid oxidation-reduction and major radical stabilization. Protein reengineering, solution NMR spectroscopy, (very) high potential protein voltammetry, quantum chemical methods, time-resolved laser spectroscopy, and protein hydrogen exchange (HX) methods will be employed to characterize and refine the redox properties of the a3X proteins. This system will be developed along three connected paths. First, the a3X proteins will be used to generate a unique protein-based thermodynamic ladder for interpreting the thermodynamic and kinetic effects of mutations and chemical modifications of amino-acid radicals in natural systems. Second, this project seeks to complement and significantly extend prior solution studies by characterizing tyrosine/phenol-based proton-coupled electron transfer in a structured protein environment. Third, we will investigate how the dynamic properties of the protein ensemble may influence tyrosine radical formation, stabilization and decay.